Identification of a key water molecule involved in the macrophage migration inhibitory factor‐catalyzed tautomerization of para‐hydroxyphenylpyruvate using neutron crystallography
Author(s)
Type
Journal Article
Abstract
Neutron crystallography was used to determine a 2.5-Å resolution all-atom structure of macrophage migration inhibitory factor (MIF) interacting with 3-(4-hydroxyphenyl)-pyruvate (HPP). MIF is a pro-inflammatory, pro-tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF-catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction-derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine-32 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N-terminal Pro-1 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room-temperature synchrotron x-ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation-induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.
Date Issued
2026-09-01
Date Acceptance
2026-05-22
Citation
Protein Science, 2026, 35 (9)
ISSN
0961-8368
Publisher
Wiley
Journal / Book Title
Protein Science
Volume
35
Issue
9
Copyright Statement
© 2026 Oak Ridge National Laboratory and The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
License URL
Publication Status
Published
Article Number
e70666
Date Publish Online
2026-08-08